The goal of this application is to investigate the roles of miRNAs in the regulation of gene expression in mammalian cells. Although at least 135 miRNAs have been identified in Caenorhabditis elegans, Drosophila melanogaster and humans, a functional role has been determined for only two: lin-4 and let-7 of C. elegans. Both of these miRNAs interact by incomplete pairing with the 3' noncoding regions of their target mRNAs. It was demonstrated that the C. elegans-specific lin-4 miRNA inhibits translation of its target mRNA, lin-14. Although the expression of the highly conserved let-7 is known to reduce the accumulation of the lin-41 gene product in C. elegans, neither the mechanism of this inhibition nor the target mRNA of let-7 orthologues in other species are known. To investigate the function of let-7 in human cells, we will isolate its human target mRNAs by expressing biotin-tagged let-7 RNA in cells and isolating target mRNAs by affinity chromatography. After obtaining cDNA copies of the target mRNAs, the nucleotides that participate in let-7/miRNA interactions will be determined by psoralen crosslinking and site-directed mutagenesis. To study the physiological roles of let7-mRNA complexes, the stability and translational efficiency of mRNAs containing wildtype and mutated binding sites for let-7 will be examined in cultured cells. To gain an understanding of the prevalence of miRNAs that regulate mRNA translation, we will search for miRNAs that are associated with polysomes in cycling cells and in mitotically arrested cells using cDNA microarrays. Identities of associated miRNAs will be determined by obtaining cDNA copies and analyzing their sequence. Using several different approaches, the experiments described in this proposal will address the function of the highly conserved miRNA let-7 and will identify distinct miRNA/mRNA complexes that are regulated during the cell cycle. If the hypothesis is correct that miRNAs can facilitate a rapid and reversible control of translation, then the proposed experiments should provide a large amount of novel biological information.The health-relatedness of this project lies in the potential roles of this novel mechanism of gene regulation in cell growth or development.